Myoregulin (MRLN, MLN) is a 46-amino-acid single-pass transmembrane micropeptide encoded by a short open reading frame in exon 3 of a skeletal-muscle transcript that was originally catalogued as a long noncoding RNA (LINC00948). It is a member of the regulin (phospholamban/sarcolipin) family of SERCA regulatory subunits: a type II membrane helix with a short cytosolic N-terminus and a few lumenal C-terminal residues, anchored in the sarcoplasmic reticulum membrane of skeletal muscle. Its transmembrane helix carries the hydrophobic motif shared with phospholamban and sarcolipin, through which it binds the SERCA calcium pump (SERCA1 predominantly, but also SERCA2a/2b in cells) as a monomer and inhibits pump activity, reducing calcium re-uptake into the sarcoplasmic reticulum. Whether it acts by lowering the pump's apparent calcium affinity or its maximal rate is debated. Myoregulin is expressed in all adult skeletal muscles and is transcriptionally controlled by MyoD/MEF2 and repressed by NR1D1. Loss of myoregulin in mice raises sarcoplasmic reticulum calcium and improves exercise performance. Knockdown in human myotubes and in a human cardiomyocyte line likewise increases SERCA activity or SR calcium content.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004857 enzyme inhibitor activity | IEA GO_REF:0000107 | MODIFY | Summary: Ensembl Compara transfer from mouse myoregulin (Q9CV60), whose UniProt IDA annotation for this term rests on SERCA Ca-ATPase assays in PMID:25640239. The human and mouse peptides are 1:1 orthologs with the SERCA-binding residues conserved, and knockdown of endogenous MLN in human cells increases SERCA activity. Reason: The inhibitory activity is real, but the term is too general and a loose fit. MLN inhibits one specific target, the SERCA calcium pump, and in current GO the P-type calcium transporter activity of SERCA (GO:0005388) is classified under transporter and ATP-dependent activity rather than catalytic activity, so "reduces a catalytic activity" describes it only indirectly. GO:0042030 ATPase inhibitor activity is directly supported by the ATPase-activity assays with MLN, and is the term curators use for the paralogous regulin PLN (IDA and IBA in human; the MF of Pln in the mouse GO-CAM model 62f58d8800005094). Proposed replacements: ATPase inhibitor activity Supporting Evidence: PMID:25640239 Together, these findings demonstrate that MLN inhibits SERCA pump kinetics similar to PLN and SLN. PMID:34445594 Using a membrane reconstitution system, we have found that MLN selectively alters the Vmax of SERCA with no effect on the KCa of SERCA (Figure 5B and Table 1). |
| GO:0004857 enzyme inhibitor activity | ISS GO_REF:0000024 | MODIFY | Summary: UniProt curator transfer from mouse myoregulin; same claim as the Ensembl IEA row. Human-cell data now independently support SERCA inhibition by endogenous human MLN (PMID:41348974, PMID:35917173). Reason: Correct in essence but too general. Replace with GO:0042030 ATPase inhibitor activity, which names the kind of activity inhibited, matches the ATPase assays used to demonstrate it (PMID:25640239, PMID:34445594, PMID:37918638), and is consistent with how the paralog PLN is annotated. Proposed replacements: ATPase inhibitor activity Supporting Evidence: PMID:25640239 MLN interacts directly with SERCA and impedes Ca(2+) uptake into the SR. PMID:37918638 replacing Lys27 with Asn significantly enhances the inhibitory potency of MLN PMID:41348974 SERCA2a activity was significantly elevated upon MLN knockdown (Figure 5A) |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Compara transfer of the mouse IDA (PMID:27923914) in which tagged regulins, including MLN, co-localised with SERCA in reticular ER membranes of transfected non-muscle cells. Reason: Not wrong (the sarcoplasmic reticulum membrane is a part of the ER membrane in GO), but the ER-membrane observation comes from heterologous expression in non-muscle cells. MRLN is a skeletal-muscle peptide, and GO:0033017 sarcoplasmic reticulum membrane, also annotated, is the physiologically informative location. Supporting Evidence: PMID:27923914 Similar to MLN, PLN, and SLN, ALN and ELN perfectly colocalized with mCherry-SERCA2b in a pattern consistent with the reticulated membranes of the ER |
| GO:0033017 sarcoplasmic reticulum membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Combined electronic annotation (orthology plus UniProt subcellular location SL-0241) placing myoregulin in the SR membrane, where mouse MLN was imaged co-localising with SERCA1 in adult muscle fibres. Reason: Correct and specific location, supported by in vivo imaging and fractionation of the mouse ortholog and by the conserved transmembrane helix. Supporting Evidence: PMID:25640239 The GFP-MLN fusion protein localized in a repeating pattern that alternated with the myosin A band and overlapped with the localization of an mCherry-SERCA1 fusion protein within the SR |
| GO:0033017 sarcoplasmic reticulum membrane | ISS GO_REF:0000024 | ACCEPT | Summary: UniProt curator transfer from mouse Q9CV60, which has an IDA for SR membrane from PMID:25640239. Reason: Core location of this SERCA regulatory peptide. No human localisation study exists, but sequence conservation of the whole transmembrane helix makes the transfer sound. Supporting Evidence: PMID:25640239 altogether suggesting that MLN functions in the membrane of the SR |
| GO:1902081 negative regulation of calcium ion import into sarcoplasmic reticulum | ISS GO_REF:0000024 | ACCEPT | Summary: Transfer of the mouse IDA/IMP annotations from PMID:25640239. MLN overexpression lowers SR Ca2+, and MLN knockout raises SR Ca2+ in myoblasts. In human cells, MLN overexpression reduces and MLN shRNA restores SR calcium content in myotubes (PMID:35917173). Reason: MLN itself performs the regulatory step, by binding the pump that imports calcium into the SR and inhibiting it, so this is direct participation in the regulation and not a downstream effect. It is the process-level counterpart of the ATPase inhibitor activity and is the most informative BP for this gene. Supporting Evidence: PMID:25640239 Strikingly, MLN KO myoblasts showed significantly increased SR Ca2+ levels compared to WT myoblasts, measured as peak SR Ca2+ release (Figure 6G). PMID:35917173 Whereas MLN overexpression in healthy myotubes reduced SR calcium content (Supplemental Figure 2, I and J), shRNA against MLN improved SR calcium content in DMD immortalized myotubes (Supplemental Figure 2, K and L). |
| GO:1901895 negative regulation of ATPase-coupled calcium transmembrane transporter activity | ISS GO_REF:0000024 | NEW | Summary: Transfer from mouse myoregulin (Q9CV60), which carries this term by IDA (PMID:27923914). Knockdown of endogenous MLN in the human AC16 cardiomyocyte line, which increases SERCA2a activity, and the reconstitution experiments corroborate it. Reason: Participation test: MLN is the entity that performs the regulatory step, binding SERCA as a monomer in the membrane and lowering its activity, so it is not merely required for the process. Comparator check: the two regulins in the same role, sarcolipin (SLN, O00631, IDA PMID:28890335) and phospholamban (PLN, P26678, IDA PMID:26673394), both carry GO:1901895 by experiment in human. This term is not an ancestor or descendant of GO:1902081, and it states the direct effect on the pump rather than the net Ca2+ flux into the SR. The only human evidence (PMID:41348974) comes from a transformed cardiac line that does not reflect MLN's normal skeletal-muscle context and is graded LOW_QUALITY below, so the annotation is coded as ISS from mouse Q9CV60 (IDA, PMID:27923914; human and mouse MLN conserve the SERCA-binding residues), with the AC16 data kept only as corroboration. Supporting Evidence: PMID:41348974 MLN knockdown significantly increased basal SERCA2a activity and abolished SFRP2-induced suppression. PMID:31449798 it was the monomeric form of all micropeptides that interacted with SERCA |
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Download this section (compressed HTML)Q: Does myoregulin inhibit SERCA by lowering its apparent Ca2+ affinity (KCa), as in the original HEK293 homogenate assays (PMID:25640239), or by lowering Vmax, as in proteoliposome co-reconstitution (PMID:34445594, PMID:37014032, PMID:37918638)? The UniProt FUNCTION text asserts the KCa mechanism, which the reconstitution data contradict.
Suggested experts: Howard S Young, L Michel Espinoza-Fonseca, Seth L Robia
Q: Should SERCA-regulin inhibition be captured as GO:0042030 ATPase inhibitor activity (as used for PLN by IBA and in GO-CAM) or as GO:0141110 transporter inhibitor activity (also carried by PLN, IDA)? In current GO, P-type calcium transporter activity is not a descendant of catalytic activity, so enzyme inhibitor activity is a poor parent for all regulins, and a consistent choice across PLN, SLN, MRLN, ERLN and ALN would help.
Q: Is myoregulin protein present at physiologically meaningful levels in human skeletal muscle fibre types, and in human heart (mRNA reported in PMID:38246425)? No human study has yet localised the endogenous peptide.
Experiment: Co-reconstitute synthetic human MRLN (and mouse Mrln side by side) with purified SERCA1a and SERCA2a in proteoliposomes, and measure Ca2+-dependent ATPase activity and Ca2+ transport to determine KCa and Vmax. Include the L29A/F30A/F33A binding-motif mutant as a negative control.
Hypothesis: Human myoregulin inhibits human SERCA1a by the same kinetic mechanism as the mouse peptide.
Type: in vitro reconstitution enzyme kinetics
Experiment: Knock out MRLN in human iPSC-derived or primary myotubes (not only DMD lines). Measure SR Ca2+ content, Ca2+ transient decay kinetics and SERCA activity, and localise epitope-tagged endogenous MRLN by knock-in.
Hypothesis: Endogenous MRLN restrains SERCA activity in human skeletal muscle.
Type: CRISPR knockout and calcium imaging
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